Interpretive Summary: Salmonella enterica subspecies enterica serotype Enteritidis (S. Enteritidis), is currently the leading cause of salmonellosis worldwide and the second leading cause in the U.S. (2, 15, 18). This serotype is a challenge to pathogen-reduction strategies, because it is capable of colonizing the avian reproductive tract, influencing oviduct physiology, and subsequently contaminating intact eggs produced by otherwise healthy hens (39, 40, 43). While it is known that non-human pathogenic Salmonella enterica serotypes Pullorum (S. Pullorum) and Gallinarum (S. Gallinarum) also contaminate the internal contents of eggs (47, 54), sporadic reports of human pathogenic serotypes Heidelberg and Typhimurium in eggs are emerging (3, 22). Thus, there is a need to develop new approaches for detecting evolution occurring within the Salmonellae that correlates with the ability to contaminate eggs.
Genome-based methods used to analyze the Salmonellae genome include pulsed- field gel electrophoresis (49), amplified fragment length polymorphism (34), multilocus sequence typing (13, 48), multiple-locus variable-number tandem-repeats (37), and ribotyping (35, 36). Although these methods detect some genetic heterogeneity within serotype, they are not sensitive enough to correlate specific biological properties, such as host range or growth characteristics, with a genotype. In order to link biological characteristics with genomic content, it is necessary to identify small scale genetic events such as single nucleotide polymorphisms (SNPs) that maintain a strong correlation to defined phenotypic traits.
This laboratory focuses on identification of small scale genetic change within Salmonella enterica that correlates with increased outbreak potential in humans. Out of all of the pathogenic Salmonella serotypes, S. Enteritidis is ideal for investigating small scale genetic events, because it generates clonally related subpopulations that vary in their ability to grow to high cell density, to produce cell surface structures, and to contaminate eggs (35). Evolution of reproductive tract tropism and the subsequent ability to contaminate eggs are the most important phenotypic characteristics for which we are pursuing genetic markers that impact human health; however, many individual metabolic differences have been identified to exist between subpopulations (42). Larger scale evolutionary events occur within the Salmonellae and may also influence outbreak 5
potential (10, 12, 19, 33, 53), but these have not been demonstrated to correlate with egg contamination. Genetic markers that correlate nearly similar genomes with variant pathogenic potential, or pathotype, are needed to improve epidemiological monitoring.
Previous investigations focusing on the intergenic spacer regions (ISR) within rRNA operons that encode ribosomal proteins achieved discrimination (8, 20, 51). In contrast, the rRNA genes themselves are too highly conserved to be useful for reliable discrimination between Salmonella serotypes that belong to the same genus and species (25). We hypothesize that the non-coding ISRs adjacent to the ribosomal genes are good candidates for correlation of genotype to phenotype, because they are not subject to the same stringent selection pressure that is required to maintain the function of the ribosomal proteins (56). In this model, the genes encoding ribosomal proteins are rooted on an undefined evolutionary time scale as relatively invariant sequence, whereas the ISR non-coding regions accumulate genetic variation somewhat faster. We hypothesize that a difference in the relative accumulation of genetic change occurs because there are evolutionary constraints on change within the ribosomal proteins, which as a group have stringent structural requirements for maintaining optimal function (27, 30, 32). There are probably limits to th

Technical Abstract:
Two intergenic spacer regions (ISRs), defined as the 179bp between the end of the 23S rrlH ORF and the start of the 5S rrfH ORF (ISR 1) and the 190bp between the end of the rrfH ORF and start of the transfer RNA aspU ORF (ISR 2) of Salmonella enterica serotype Enteritidis, were sequenced from 64 isolates that included 6 different pathogenic Salmonella enterica group serotypes, namely Typhimurium, Pullorum, Gallinarum, Heidelberg, Newport, and Enteritidis. Evolutionary divergence between the two serogroups D1 (Pullorum, Gallinarum, and Enteritidis) and B (Typhimurium and Heidelberg) was found in ISR 1. Newport, a serotype C1 Salmonellae, formed a third clade that included similarity to the human specific pathogens, Paratyphi (A) and Typhi (D1). Results for ISR 2 showed that serotype Heidelberg shares evolutionary traits with serogroup D1 serotypes that are associated with egg contamination. ISR 2 sequence for Salmonella Pullorum further suggests that a past insertion event may have resulted in different evolutionary paths for the Salmonella serotypes examined here, one of which is generally associated with avian adaptation and reproductive tract tropism. In summary, these results suggest that the sequences of the rrlH-rrfH and rrfH-aspU ISRs are useful markers for following evolutionary trends within Salmonella enterica that substantially impact human health.